Manipulation of the rate of gastrointestinal transit by modulating intestinal methane concertration

ABSTRACT

Disclosed is a method of manipulating the rate of gastrointestinal transit in a mammalian subject Also disclosed is the use, in the manufacture of a medicamens for the treatment of constipation, of a selective inhibitor of methanogensis, a methanogen-displacing probiotic agent, or a prebiotic agent that inhibits the growth of methanogenic bacteria or promotes the growth of competing non-methanogenic intestinal flora. Alternatively, in accordance with the invention, is disclosed the use in the manufacture of a medicament for the treatment of diarrhea, of methane, or a methane precursor, a methanogenic or other methane-enhancing probiotic agent, or a methanogenesis-enhancing prebiotic agent.

BACKGROUND OF THE INVENTION Discussion of the Related Art

Irritable bowel syndrome (IBS) is a common gastrointestinal disorderseen in more than 15% of the population (1,2).

Over the last few years, progress has been made in characterizingirritable bowel syndrome (IBS). Studies have demonstrated altered gutmotility (3), peripheral (4) and central (5) sensory dysfunction, aswell as an exaggerated response to stress (6) in this syndrome. However,there is no finding that can be identified in a majority of patients andby extension, there is no diagnostic test that is associated with IBS.As a result, investigators have created complex diagnostic schema suchas the Rome criteria to help diagnose and categorize the syndrome (7,8).

One consistent clinical finding in IBS is gas in combination withbloating and visible distention (9,10). Koide et al. recently foundsmall intestinal gas to be significantly increased in IBS compared tocontrols (11) regardless of whether subjects conform to diarrhea,constipation or pain subgroups.

Excessive small intestinal gas can occur as a result of increasedproduction of gas within the gut by bacterial fermentation. Hydrogen andmethane are common gases excreted during breath testing (43). Althoughhydrogen production appears more ubiquitous, methane production is seenin 36-50% of healthy subjects (27, 41, 42). In particular, methane isnoted to be common in diverticulosis (25), and less prevalent indiarrheal conditions such as Crohn's or ulcerative colitis (26-28).Recent data suggests that children with encopresis have excessive breathmethane on lactulose breath test (“LBT”; 13). This finding has not beenextended to adults with constipation-predominant IBS.

A condition known to produce excessive small bowel gas is smallintestinal bacterial overgrowth (SIBO). Small intestinal bacterialovergrowth is a condition in which the small bowel is colonized byexcessive amounts of upper or lower gastrointestinal tract flora.Although there are many conditions associated with SIBO, recent studieshave demonstrated an increased prevalence of SIBO in irritable bowelsyndrome (IBS) (12) and it is a recognized cause of diarrhea ininflammatory bowel disease (IBD) (28, 39, 40).

There is some support for the association between altered breath testingresults and enteric flora in IBS. In one study, 56% of diarrheapredominant IBS subjects were found to have a positive ¹³C-xylose breathtest (20). In another study, flagyl was reported to be superior toplacebo in reducing clinical symptoms in IBS (21). The authors in thatpaper were uncertain of the mechanism for this improvement.

One method of diagnosing SIBO is the lactulose breath test (LBT) whereovergrowth is considered to be present if a greater than 20 ppm rise inbreath hydrogen or methane concentration is observed within 90 minutesof oral administration of lactulose (19).

In a recent study, we suggested that a large percent (78%) of IBSsubjects has SIBO as diagnosed by lactulose breath test (12). Someworkers criticize the reliability of LBT to diagnose SIBO since in theidentification of any infectious process, culture is the gold standard.The main issue with culture is accessibility. Riordan, et al. comparedbreath testing to direct culture and found the breath test to lackreliability (29). This and other similar studies were confounded bytheir selection of subjects who had surgically altered anatomypredisposing to the development of upper GI tract. SIBO. Since SIBO (insurgically naive patients) is often an expansion of colonic bacteria,the direction of expansion is retrograde involving first the distalsmall intestine. As such, direct culture is only practical in thepatient whose SIBO is so severe that the bacteria has expandedproximally into the duodenum or proximal jejunum.

Regardless of some skepticism about the reliability of LBT to diagnoseSIBO, there are similarities between SIBO and IBS. Bloating, a featureof SIBO, is also classically associated with IBS (10). In SIBO, bloatingis due to small intestinal fermentation of nutrients. Until recently,gas studies in IBS have been limited to the investigation of flatus.Yet, even these studies suggest the presence of excessive bacteria inIBS. King, et al found the production of hydrogen by IBS subjects to befive-fold elevated implying excessive enteric bacteria (22). Recently,data suggest that IBS patients have excessive gas and that this gas islocalized to the small intestine (11). However, the contrasting diarrheaand constipation predominant subgroups in IBS remain unexplained.

The speed of transit through the small intestine is normally regulatedby inhibitory mechanisms located in the proximal and distal smallintestine known as the jejunal brake and the ileal brake. Inhibitoryfeedback is activated to slow transit when end products of digestionmake contact with nutrient sensors of the small intestine. (E.g., Lin,H. C., U.S. Pat. No. 5,977,175; Dobson, C. L. et al., The effect ofoleic acid on the human ileal brake and its implications for smallintestinal transit of tablet formulations, Pharm. Res. 16(1):92-96[1999]; Lin, H. C. et al., Intestinal transit is more potently inhibitedby fat in the distal (Ileal brake) than in the proximal (jejunal brake)gut, Dig. Dis. Sci. 42(1):19-25 [1997]; Lin, H. C. et al, Jejunal brake:inhibition of intestinal transit by fat in the proximal small intestine,Dig. Dis. Sci, 41(2):326-29 [1996a]).

Methane in the intestinal lumen has never before been reported to affectthe rate of gastrointestinal transit.

SUMMARY OF THE INVENTION

The present invention relates to a method of manipulating the rate ofgastrointestinal transit in a mammalian subject, including a humanpatient. The method involves: (a) increasing the rate ofgastrointestinal transit by causing the partial pressure of methane inthe subject's intestines to be decreased; and (b) decreasing the rate ofgastrointestinal transit by causing the partial pressure of methane inthe subject's intestines, for example in the distal gut, to beincreased.

Thus, by practicing the inventive method to increase the rate ofgastrointestinal transit, constipation and disorders exhibitingconstipation can be treated in subjects in whom abnormally elevatedintestinal methane levels are detectable (e.g., in cases ofconstipation-predominant irritable bowel syndrome [IBS],pseudoobstruction, colonic inertia, postoperative ileus, encopresis,hepatic encephalopathy, or medication-induced constipation). Inaccordance with this embodiment of the present invention, the partialpressure of methane in the subject's intestines can be decreased byadministering to the subject's intestinal lumen a selective inhibitor ofmethanogenesis, such as monensin, or a methanogen-displacing probioticagent, or a prebiotic agent that inhibits the growth of methanogenicbacteria or promotes the growth of competing non-methanogenic intestinalflora.

Consequently, the present invention is also directed to the use in themanufacture of a medicament for the treatment of constipation, of aselective inhibitor of methanogenesis, or of a methanogen-displacingprobiotic agent, or of a prebiotic agent that inhibits the growth ofmethanogenic bacteria or promotes the growth of competingnon-methanogenic intestinal flora.

And alternatively, by practicing the inventive method to decrease therate of gastrointestinal transit, patients with diarrhea and disordersexhibiting diarrhea can be treated (e.g., cases of diarrhea-predominantIBS, Crohn's disease, ulcerative colitis, celiac disease, microscopiccolitis, dumping syndrome, rapid transit, short bowel syndrome,post-gastrectomy syndrome, diabetic diarrhea, hyperemesis, orantibiotic-associated diarrhea). In accordance with this embodiment ofthe present invention, the partial pressure of methane in the subject'sintestines can be increased by administering methane gas to theintestinal lumen of the subject, for example into the distal segment ofthe intestine of the subject, or by administering to the subject amethanogenic probiotic agent or methogenesis-enhancing prebiotic agent.

Consequently, the present invention is also directed to the use in themanufacture of a medicament for the treatment of diarrhea, of methane ora methane precursor, or of a methanogenic or other methane-enhancingprobiotic agent, or of a methogenesis-enhancing prebiotic agent.

These and other advantages and features of the present invention will bedescribed more fully in a detailed description of the preferredembodiments which follows.

BRIEF DESCRIPTION OF TIE DRAWINGS

FIG. 1 shows a patient flow chart for a double-blind, randomized,placebo-controlled study confirming that an abnormal lactulose breathtest is more prevalent in IBS than normal controls, and that antibiotictreatment in IBS leads to an improvement in symptoms and that this isbased on antibiotic-induced normalization of breath test.

FIG. 2 shows percent improvement in composite score based on treatmentand success in normalizing the LHBT. Data=mean % reduction in compositescore; the difference in the composite score was significant (p=0.01,1-way ANOVA). The difference in patient reported improvement was alsosignificant (p<0.000001, 1-way ANOVA). In the neomycin treated groups,the data were analyzed according to success of treatment. Neo=Neomycin.

FIG. 3 shows a comparison of percent reported bowel normalizationbetween and within gender groups; NS=not significant.

FIG. 4 show the pattern of gas production with IBS symptom type, i.e.,constipation-predominant IBS (unshaded bars; p<0.00001) versusdiarrhea-predominant IBS (shaded bars; p<0.001).

FIG. 5 show the pattern of gas production in IBS patients (n=65) withrespect to symptom severity in those with constipation-predominant IBS(unshaded bars; p<0.00001) versus those with diarrhea-predominant IBS(shaded bars; p<0.00001).

FIG. 6 illustrates the effect on intestinal transit in dogs administered180 ml of room air (circles) or methane gas (squares) by bolus deliveryto the distal gut. Methane slowed intestinal transit.

FIG. 7 shows mean diarrhea and constipation severity scores of allsubjects (n=551) with SIBO as a function of the of type of gas patternproduced on LBT; p<0.00001 for trend in reduction of diarrhea with thepresence of methane (one-way ANOVA); p<0.05 for the trend towardsincreasing constipation with the presence of methane (one-way ANOVA).

FIG. 8 shows mean diarrhea and constipation severity scores of IBSsubjects (n=296) with SIBO as a function of the of type of gas patternproduced on LBT; p<0.001 for trend in reduction of diarrhea with thepresence of methane (one-way ANOVA); p<0.05 for the trend towardsincreasing constipation with the presence of methane (one-way ANOVA).

FIG. 9 shows mean constipation minus diarrhea (C-D) severity score forthe whole group (n=551) and IBS subjects (n=296) as a function of thetype of gas pattern produced on LBT; p<0.00001 for trend in C-D forwhole group (one-way ANOVA); p<0.0001 for trend in C-D for IBS subjects(one-way ANOVA).

FIG. 10 shows the percentage of IBS subjects (n=296) exhibiting each gaspattern who reported constipation vs. diarrhea predominant symptoms.

FIG. 11 shows the percentage of subjects with IBD who produced each ofthe three abnormal gas patterns on LBT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, the partial pressure ofmethane in the subject's intestines can be decreased by administering tothe subject's intestinal lumen a selective inhibitor of methanogenesis,such as monensim Useful selective inhibitors of methanogenesis includeHMG-CoA reductase inhibitors known in the art (e.g., U.S. Pat. No.5,985,907) that selectively inhibit the growth of methanogenic bacteriawithout significantly inhibiting the growth of non-methanogens, forexample in the distal gut or colon of the subject.

Alternatively, or concurrently, in accordance with the presentinvention, the partial pressure of methane in the subject's intestinescan be decreased by administering to the subject's intestinal lumen amethanogen-displacing probiotic agent to inhibit the growth ofmethanogenic bacteria therein, for example, an inoculum of a lactic acidbacterium, bifidobacterium, or probiotic Saccharomyces species, e.g., S.cerevisiae. (A. S. Naidu et al., Probiotic spectra of lactic acidbacteria, Crit. Rev. Food Sci. Nutr. 39(1):13-126 [1999]; J. A.Vanderhoof et al. [1998]; G. W. Tannock, Probiotic propertyies of lacticacid bacteria: plenty of scope for R & D, Trends Biotechnol.15(7):270-74 [1997]; S. Salminen et al., Clinical uses of probiotics forstabilizing the gut mucosal barrier: successful strains and futurechallenges, Antonie Van Leeuwenhoek 70(2-4):347-58 [1997]; ChaucheyrasF. et al, In vitro H ₂ utilization by a ruminal acetogenic bacteriumcultivated alone or in association with an archaea methanogen isstimulated by a probiotic strain of Saccharomyces cerevisiae, ApplEnviron Microbiol 61(9):3466-7 [1995]). The inoculum is typicallyadministered in a pharmaceutically acceptable ingestible formulation,such as in a capsule, or for some subjects, consuming a foodsupplemented with the inoculum is effective, for example a milk,yoghurt, cheese, meat or other fermentable food preparation Usefulprobiotic agents include Bifidobacterium sp. or Lactobacillus species orstrains, e.g., L. acidophilus, L. rhamnosus, L. plantarum, L. reuteri,L. paracasei subsp. paracasei, or L. casei Shirota, (P. Kontula et al.,The effect of lactose derivatives on intestinal lactic acid bacteria, J.Dairy Sci. 82(2):249-56 [1999]; M. Alander et al., The effect ofprobiotic strains on the microbiota of the Simulator of the HumanIntestinal Microbial Ecosystem (SHIME), Int. J. Food Microbiol.46(l):71-79 [1999]; S. Spanhaak et al., The effect of consumption ofmilk fermented by Lactobacillus casei strain Shirota on the intestinalmicroflora and immune parameters in humans, Eur. J. Clin Nutr.52(12):899-907 [1998]; W. P. Charteris et at, Antibiotic susceptibilityof potentially probiotic Lactobacillus species, J. Food Prot.61(12):1636-43 [1998]; B. W. Wolf et at, Safety and tolerance ofLactobacillus reuteri supplementaton to a population infected with thehuman immunodeficiency virus, Food Chem. Toxicol. 36(12):1085-94 [1998];G. Gardiner et al., Development of a probiotic cheddar cheese containinghuman-derived Lactobacillus paracasei strains, Appl. Environ Microbiol.64(6):2192-99 [1998); T. Sameshima et al., Effect of intestinalLactobacillus starter cultures on the behaviour of Staphylococcus aureusin fermented sausage, Int. J. Food Microbiol. 41(1):1-7 [1998]).

Alternatively, or concurrently, in accordance with the presentinvention, the partial pressure of methane in the subject's intestinescan be decreased by administering to the subject's intestinal lumen aprebiotic agent that inhibits the growth of methanogenic bacteria orpromotes the growth of competing non-methanogenic intestinal flora.(E.g., Tuohy K M et al., The prebiotic effects of biscuits containingpartially hydrolysed guar gum and fructo-oligosaccharides—a humanvolunteer study, Br J Nutr 86(3):341-8 [2001]).

In accordance with the present invention, the partial pressure ofmethane in the subject's intestines can be increased by administeringmethane to the subject's intestinal lumen. Accordingly, methane can beadministered directly to the intestine by infusion through a tube,preferably via the rectum, but other access routes for intubation to theintestine are also useful. Alternatively, methane can be administered tothe intestinal lumen by providing a medicament comprising a catalyst andchemical substrate (i.e., a “methane precursor”) to the intestinallumen, where they come in contact to produce methane in situ. Forexample, the catalyst and substrate can be administered in separatecontrol release tablets, which release their contents in the desiredlocation in the intestine.

Alternatively, in accordance with the present invention, the partialpressure of methane in the subject's intestines can be increased byadministering to the subject's intestinal lumen a methane-enhancingprobiotic agent. A “methane-enhancing” probiotic agent is one thateffectively enhances the partial pressure of methane in the subject'sintestinal lumen. The methane enhancing probiotic agent can be amethanogenic bacterium, such as Methanobrevibacter smithii, or certainBacteroides spp. or Clostridium spp. (see, e.g., McKay L. F et al.,Methane and hydrogen production by human intestinal anaerobic bacteria,Acta Pathol Microbiol Immunol Scand [B] 90(3):257-60 [1982]), or anorganism that can enhance the growth of intestinal methanogens, such asClostridium butyricum.

Alternatively, or concurrently, in accordance with the presentinvention, the partial pressure of methane in the subject's intestinescan be increased by administering to the subject's intestinal lumen aprebiotic agent that enhances the growth of methanogenic bacteria.

As the term is commonly used in the art, the “proximal” segment of thesmall bowel, or “proximal gut”, comprises approximately the first halfof the small intestine from the pylorus to the mid-gut. The distalsegment, or “distal gut” includes approximately the second half, fromthe mid-gut to the ileal-cecal valve.

Representative methods of administering include giving, providing,feeding or force-feeding, dispensing, inserting, injecting, infusing,perfusing, prescribing, furnishing, treating with, taking ingesting,swallowing, eating or applying. Administration of inhibitors, probioticagents, or prebiotic agents, is by well known means, including mostpreferably oral administration and/or enteral administration.

Detection of intestinal methane and other gases, while not essential tothe practice of the invention, can be accomplished, if desired, by anysuitable means or method known in the art. For example, one preferredmethod is breath testing. (E.g., P. Kerlin and L. Wong, Breath hydrogentesting in bacterial overgrowth of the small intestine, Gastroenterol.95(4):982-88 [1988]; A Strocchi et al., Detection of malabsorption oflow doses of carbohydrate: accuracy of various breath H ₂ criteria,Gastroenterol. 105(5):1404-1410 [1993]; D. de Boissieu et al., [1996];P. J. Lewindon et al., Bowel dysfunction in cystic fibrosis: importanceof breath testing, J. Paedatr. Child Health 34(1):79-82 [1998]). Breathhydrogen or breath methane tests are based on the fact that manyobligately or facultatively fermentative bacteria found in thegastrointestinal tract produce detectable quantities of hydrogen ormethane gas as fermentation products from a substrate consumed by thehost, under certain circumstances. Substrates include sugars such aslactulose, xylose, lactose, sucrose, or glucose. The hydrogen or methaneproduced in the small intestine then enters the blood stream of the hostand are gradually exhaled.

Typically, after an overnight fast, the patient swallows a controlledquantity of a sugar, such as lactulose, xylose, lactose, or glucose, andbreath samples are taken at frequent time intervals, typically every 10to 15 minutes for a two- to four-hour period. Samples are analyzed bygas chromatography or by other suitable techniques, singly or incombination. A variable fraction of the population fails to exhaleappreciable hydrogen gas during intestinal fermentation of lactulose;the intestinal microflora of these individuals instead produce moremethane. (G. Corazza et al, Prevalence and consistency of low breath H ₂excretion following lactulose ingestion. Possible implications for theclinical use of the H ₂ breath test, Dig. Dis. Sci. 38(11):2010-16[1993); S. M. Riordan et al, The lactulose breath hydrogen test andsmall intestinal bacterial overgrowth, Am. J. Gastroentrol.91(9);1795-1803 [1996)). A non-digestible substrate other than lactulosecan optionally be used.

Another useful method of detecting intestinal gases, such as methane, isby gas chromatography with mass spectrometry and/or radiation detectionto measure breath emissions of isotope-labeled carbon dioxide, methane,or hydrogen, after administering an isotope-labeled substrate that ismetabolizable by gastrointestinal bacteria but poorly digestible by thehuman host, such as lactulose, xylose, mannitol, or urea. (E.g., G. R.Swart and J. W. van den Berg, ¹³ C breath test in gastrointestinalpractice, Scand. J. Gastroenterol. [Suppl.] 225:13-18 [1998]; S. F.Dellert et al., The 13C-xylose breath test for the diagnosis of smallbowel bacterial overgrowth in children, J. Pediatr. Gastroenterol. Nutr.25(2):153-58 [1997]; C. E. King and P. P. Toskes, Breath tests in thediagnosis of small intestinal bacterial overgrowth, Crit. Rev. Lab. Sci.21(3):269-81 [1984)). A poorly digestible substrate is one for whichthere is a relative or absolute lack of capacity in a human forabsorption thereof or for enzymatic degradation or catabolism thereof.

Suitable isotopic labels include ¹³C or ¹⁴C. For measuring methane orcarbon dioxide, suitable isotopic labels can also include ²H and ³H or¹⁷O and ¹⁸O, as long as the substrate is synthesized with the isotopiclabel placed in a metabolically suitable location in the structure ofthe substrate, i.e., a location where enzymatic biodegradation byintestinal microflora results in the isotopic label being sequestered inthe gaseous product. If the isotopic label selected is a radioisotope,such as ¹⁴C, ³H or ¹⁵O, breath samples can be analyzed by gaschromatography with suitable radiation detection means. (E.g., C. S.Chang et al., Increased accuracy of the carbon-14 D-xylose breath testin detecting small-intestinal bacterial overgrowth by correction withthe gastric emptying rate, Eur. J. Nucl. Med. 22(10):1118-22 [1995]; C.E. King and P. P. Toskes, Comparison of the 1-gram ¹⁴ C]xylose, 10-gramlactulose-H ₂ , and 80-gram glucose-H ₂ breath tests in patients withsmall intestine bacterial overgrowth, Gastroenterol. 91(6):1447-51[1986]; A Schneider et al., Value of the ¹⁴ C-D-xylose breath test inpatients with intestinal bacterial overgrowth, Digestion 32(2):86-91[1985]).

The preceding are merely illustrative and non-exhaustive examples ofmethods for detecting small intestinal bacterial overgrowth.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 Methane Excretion on Breath Test has a PositivePredictive Value of 100% for Constipation-Predominant IBS

In a double-blind, randomized, placebo-controlled study, we confirm thatan abnormal lactulose breath test is more prevalent in IBS than normalcontrols, and that antibiotic treatment in IBS leads to an improvementin symptoms and that this is based on antibiotic-induced normalizationof breath test. Secondly, lactulose breath test profiles are evaluatedto show that gaseous constituents vary among IBS subgroups. In thisstudy, we show that methane excretion on breath test has a positivepredictive value of 100% for constipation predominant IBS. This isanother important step in linking SIBO and IBS.

A. Materials and Methods

Study Population

Study subjects were recruited by advertising in local newspapers, radioand IBS support groups throughout the greater Los Angeles are& To avoidreferral bias, subjects were not recruited through the GI motilityclinic or any gastroenterology practice based at Cedars-Sinai MedicalCenter. Subjects were included if they met Rome I criteria for IBS (7).Rome I was chosen as it does not prejudice between diarrhea andconstipation, and no peer-review publications were available to validateRome II as a diagnostic strategy (14). Subjects were excluded if theyhad antibiotics within the previous three months, a previous lactulosebreath test (LBT), or a history of diabetes, thyroid disease, intestinalsurgery (except cholecystectomy or appendectomy), connective tissuedisease, narcotic use or known gastrointestinal disease. Subjects withrenal insufficiency, hearing impairment, probiotic use or allergy toaminoglycosides were also excluded. Approval from the institutionalreview board and written informed consent from the participatingsubjects were obtained.

In an initial comparison, 15 sex-matched normal controls were identifiedbased on the absence of all Rome I criteria. These subjects underwentlactulose breath testing and the prevalence of abnormal breath test wascompared to subjects with IBS.

Study Design

Subjects presented to the GI Motility Laboratory having fasted from 7:00p.m. the night before. They were instructed not to ingest legumes or aheavy meal for dinner the night prior to evaluation. Good oral hygienewas recommended and smoking was not permitted on the day of testing.

Prior to the LBT, subjects completed a symptom questionnaire asking themto grade nine IBS symptoms (abdominal pain, diarrhea, constipation,bloating, sense of incomplete evacuation, straining, urgency, mucus andgas) on a severity score of 0-5 as has been previously used andrecommended (15-17). All questions were answered based on their recallof the preceding 7 days (17).

Subjects then underwent a LBT by ingesting 10 g of lactulose (InalcoSpa, Milano, Italy, packaged by Xactdose Inc., South Beloit, Ill.)followed by 1-2 ounces of water after an initial baseline breath sample.Breath samples were then collected at 15 minute intervals for 180minutes. End expiratory breath samples were taken to ensure alveolar gassampling. Samples were analyzed for hydrogen, methane and carbon dioxideusing a Model SC, Quintron gas chromatograph (Quintron InstrumentCompany, Milwaukee, Wis.). Carbon dioxide measurements were used tocorrect for the quality of alveolar sampling. Measurements were plottedgraphically as previously described (12). Patients and investigatorswere blinded to the result of the breath test.

All subjects were randomized by personnel not associated with the studyto receive, in a double blind fashion, either neomycin (500 mg) (Tevapharmaceuticals, USA, Sellersville, Pa.) or matching placebo twice dailyfor 10 days. Seven days after completion of the antibiotic or placebo,subjects returned for a repeat questionnaire and LBT. A seven day followup was chosen since in our experience the abnormal breath test in IBScan recur as early as two weeks after antibiotic normalization. As partof the follow up questionnaire, subjects were asked to subjectively ratethe amount of improvement they experienced as a percent normalization ofbowel function and repeated their perceived severity of the 9 bowelsymptoms described earlier. Compliance was assessed by pill count. Tocomply with institutional review board requirements, follow-up LBTresults could not be blinded so patients could seek appropriate medicaltherapy for their test result.

At the completion of enrollment, all initial and follow-up breath testswere coded and randomized by personnel not involved in theinterpretation of the test. A blinded reviewer (M.P.) interpreted theresults and was asked to categorize the breath tests based on whetherthe test met the criteria for normal LBT. A normal LBT was defined as,no rise of breath hydrogen (H₂) or methane (CH₄) concentration before 90minutes of lactulose, with a definitive rise never more than 20 ppmduring 180 minutes of measurement (18, 19, 37, 38). Studies that fellout of this range were categorized as abnormal. A second set of criteriafor breath test interpretation was also used whereby the traditional 2peaks to suggest bacterial overgrowth were required. Since the two peakmethod was not well not as well validated a technique (37) as the partsper million (ppm), this finding was only used to compare the prevalenceof this finding to healthy controls.

Measures of Outcome

Data were analyzed using an intention-to-treat method. The primaryoutcome measure was based on a composite score (CS) calculated from the3 main IBS symptoms (abdominal pain, diarrhea and constipation each on ascale from 0-5) to generate a score out of 15 (most severe). This wasdone to account for the severity of all potential IBS subgroups. Sinceother IBS symptoms (such as straining) would worsen or improve dependingon whether patients started with diarrhea or constipation, respectively,minor criteria were not included in the CS. In addition, as reduction incolonic organisms could result in an improvement in gas and bloating,irrespective of bacterial overgrowth, gaseous symptoms too were excludedfrom the score. The percent improvement in the CS was then comparedbetween placebo and neomycin In addition, the overall percent bowelnormalization as determined by patient reporting was likewise compared.

The prevalence of a true clinical response was then determined andcompared between placebo and neomycil A true clinical response wasdefined as a ≧50% reduction in CS. Secondarily, a true clinical responsewas also assessed based on subjects reporting their overall percentbowel normalization. A ≧50% normalization implied a true clinicalresponse. This method of analysis closely followed the multinationalconsensus recommended guidelines for data analysis in IBS clinicalstudies (16).

Secondary endpoints included a similar analysis of gender subgroups.Subsequently, IBS subgroups were identified whereby diarrhea predominantIBS was deemed present when diarrhea severity (0-5 scale) was greaterthan constipation in any individual subject. The opposite proportiondetermined constipation predominance. This means of identifying diarrheaand constipation predominant subgroups was chosen since criteria forthese subgroups are not validated and based subjectively on physicianinterview (14). This approach further reduced bias since subjects wouldnot be aware of the interest in subgrouping their predominant feature.

A post hoc analysis was then conducted on all abnormal breath testresults to determine if the type of gas produced on LBT was related toIBS subgroup. The abnormal breath tests were divided into two abnormaltest groups: hydrogen production only and any methane production. Therelationship between constipation predominant IBS and diarrheapredominant IBS to the type of gas seen was determined. Subsequently, ina more objective fashion, the severity score for diarrhea andconstipation were then compared between gas types. Finally, a scorebased on the difference between constipation and diarrhea severity(i.e., constipation score minus diarrhea score; “C-D”) was determined.The C-D was used to examine the relative weight of constipation todiarrhea in individual subjects (the more positive the score the greaterthe dominance the constipation was compared to diarrhea). Subjects withidentical score for constipation and diarrhea severity were excludedfrom these analyses. This C-D score was also compared between gas types.

Finally, to support the principal that the abnormal test in IBS was notdue to rapid transit, the mean breath test profile in constipation anddiarrhea predominant IBS was compared. Since it is suggested in theliterature that diarrhea predominant IBS is associated with rapidtransit (34-36) and constipation predominant IBS with slow transit (34,35), the hydrogen profile should be different in both groups.

Statistical Analysis

The number of subjects enrolled in the study was determined based on thedetection of a 10% difference between placebo and neomycin This furtherassumed a 15% variance and an α=0.05 with power of 90% in a 2-sidedanalysis.

Quantitative data were compared using the Student's t-test with resultsexpressed as mean±S.E. Comparisons of qualitative data utilized Fisher'sExact Test for comparison of IBS subjects to healthy controls. All othercomparisons of qualitative data utilized Chi-square. A 1-way ANOVA wasused to compare the results of the 3 groups: placebo treated, neomycinwith unsuccessful normalization of LBT and neomycin treated withsuccessful normalization of LBT.

B. Results

Subject Demographics

Two-hundred and thirty-one subjects were screened (FIG. 1). Of these,111 met enrollment criteria. However, 10 of these 111 subjects hadincomplete data (6 in neomycin group and 4 in placebo group). Thespecific reasons for incomplete data were, voluntary prematurewithdrawal (n=3), no follow up breath test (n=4), failure to show up forfollow up (n=1), no follow-up questionnaire (n=1) and prematurewithdrawal by subject due to severe diarrhea (n=1). Despite theincomplete data, these subjects were included in the intention-to-treatanalyses and they were counted as no (0%) improvement. The baselinecharacteristics were similar for the neomycin and placebo groups (Table1, below). TABLE 1 Comparison of demographics between placebo andneomycin. Characteristic Placebo Neomycin p-value n 56 55 Age 41.9 ± 0.244.7 ± 0.2 NS Sex (F/M) 27/29 34/21 NS Baseline Composite  8.7 ± 0.4 8.8 ± 0.3 NS Score Abnormal breath test [n 47 (84)  46 (84)  NS (%)]Diarrhea predominant 21 (40)> 25 (48)* NS IBS [n (%)] Constipation 20(38)> 18 (35)* NS predominant IBS [n (%)] Other IBS subgroup [n 11 (21)>7 (13)* NS (%)]Data are mean ± S.E. Baseline composite score = pain severity + diarrheascore + constipation score (each on a scale from 0-5) before treatment.Other IBS subgroup = subjects with constipation severity = diarrheaseverity.*Only 52 subjects in the neomycin group completed the questionnairesufficiently to determine this result.>Only 52 subjects in the placebo group completed the questionnairesufficiently to determine this result. NS = not significant.Case-Control Comparison

IBS subjects had a higher prevalence of abnormal LBT than sex-matchedcontrols with 93 out of 111 (84%) subjects f g these criteria comparedto 3 out of 15 (20%) sex-matched controls (OR=26.2, CI=4.7-103.9,p<0.00001). When comparing the prevalence of abnormal LBT with doublepeak, 55 out of 111 IBS subjects (50%) were positive compared to 2 outof 15 healthy controls (13%) (p=0.01).

Primary Outcome Measures

In the intention-to-treat analysis, neomycin resulted in a 35.0±0.7%reduction in CS compared to a 11.4±1.3% reduction in the placebo group(p<0.05). In the subgroup of patients with abnormal baseline LBT (n=3),neomycin produced a 35.4±0.8% reduction in CS versus a 3.7±1.6%reduction in the placebo group (p<0.01). No difference was seen insubjects with a normal baseline breath test although a higher placeborate was reported in this very small group (51%).

Ninety-one out of the 111 subjects completed their percent bowelnormalization question after treatment. Of these 91 subjects, neomycinresulted in a 40.1±5.3% reported bowel normalization compared to15.1±3.6% for placebo (p<0.001). Amongst the subgroup of subjects withabnormal initial breath tests, neomycin resulted in a 44.8±5.6%normalization compared to 11.0±3.3% for placebo (p<0.00001).

Neomycin was more likely to result in a true clinical response thanplacebo. Among all subjects receiving neomycin, 24 out of 55 (43%)experienced a ≧50% improvement in CS versus 13 out of 56 (23%) in theplacebo group (OR=4.3, CI=1.05-6.3, p<0.05). In the subgroup of subjectswith abnormal breath tests, 21 out of 46 (46%) receiving neomycin had aclinical response compared to 7 out of 47 (15%) in the placebo group(OR=4.8, CI=1.62-14.7, p<0.01). Using patient's subjective report ofpercent bowel normalization, in the whole group of subjects who answeredthis question (n=91), 50% of subjects receiving neomycin had a trueclinical response in contrast to 17% of subjects getting placebo(OR=4.8, CI=1.7-14.4, p<0.01). In those with abnormal initial breathtest, 55% of neomycin and 11% of placebo treated subjects had a trueclinical response (OR=9.6, CI=2.5-39.7, p<0.0001). Finally, 7 out of the8 subjects (88%) who had a normal follow up LBT after neomycin reportedmore than 50% normalization of bowel function.

Of the 111 subjects, only the 101 subjects with complete data were usedin the remainder of the analyses.

Of 84 out of 101 subjects with an abnormal baseline LBT, 41 were treatedwith neomycin Eight out of 41 (20%) achieved normalization of LBT. Oneout of 43 subjects in the placebo group went from an abnormal breathtest to normal. A significant difference in symptom response was seendepending on the outcome of treatment in these abnormal subjects.Specifically, the percent reduction in CS was different in the following3 groups: subjects receiving placebo (4.1±11.7%), neomycin-treated groupthat did not achieve LBT normalization (34.4±6.2%) and neomycin-treatedgroup with LBT norm zation (61.7±9.4%) (p=0.01, 1-way ANOVA) FIG. 2).Using patients self-report of percent bowel normalization, the 3 groupswere more different. Subjects receiving placebo reported 11.0±3.7%normalization, subjects receiving neomycin but not successfulnormalization of LBT, 36.7±6.1% and those subjects with normal follow upLBT after neomycin reporting 75.0±6.4% bowel normalization (p<0.0000001,1-way ANOVA).

Neomycin, although statistically more effective than placebo, was onlyable to normalize the breath test 20% of the time. This may be due tothe large numbers and types of enteric organisms (30-33) or bacterialresistance.

Transit Comparison

When the mean hydrogen breath test profile was compared between diarrheaand constipation predominant IBS subjects, there was no evidence thatdiarrhea predominance had earlier hydrogen appearance (data not shown).In fact, diarrhea and constipation profiles were both virtuallysuperimposable and not different at any time point with a mean of >20ppm at 90 minutes in both groups.

Adverse Events

One subject developed profuse watery diarrhea while taking placebo. Thecause of the diarrhea was later found to be food poisoning. Two of theenrolled subjects were found to have other diagnoses. The first subjecthad an 8 cm mass in the abdomen. The surgical specimen demonstratednon-Hodgkin's lymphoma. This subject was in the placebo group. Thesecond subject was noted to have urinary retention, which precipitatedbowel complaints. The second subject was in the neomycin group. Boththese subjects had a normal initial LBT. Both were included as part ofthe intention-to-treat analysis.

Effect of Gender

Both male and female subjects were noted to have a significantly greaterimprovement in percent bowel normalization over placebo (FIG. 3).Furthermore, there was no difference in response rate between male andfemale patients.

Type of Gas and IBS Subgroup

The type of gas produced by IBS subjects on LBT was predictive of theirsubtype of IBS amongst the 84 subjects with abnormal baseline. Afterexclusion of subjects with no gas production (n=4) and subjects whereconstipation severity was equal to diarrhea (n=15), 34 diarrheapredominant and 31 constipation predominant IBS subjects were analyzed.Twelve out of 31 constipation-predominant subjects (39%) excretedmethane whereas no methane excretion was seen in the 34 diarrheapredominant subjects (OR=∞, CI=3.7-4.3, p<0.001, positive predictivevalue=100%) (Table 2, below; and FIG. 4). The severity of constipationwas 4.1±0.3 in subjects with methane excretion but only 2.3±0.2 innon-methane excretors (p<0.01) (Table 3, below). In a similarcomparison, the C-D was 2.8±0.5 in methane excretors and −0.7±0.3 forhydrogen excretors (p<0.00001) (Table 3; and see FIG. 5). TABLE 2Comparison of IBS subgroups based on methane and hydrogen excretion withabnormal breath test. (n = 65)* Hydrogen Methane Diarrhea 34 0Constipation 19 12*After exclusion of subjects with no gas production (n = 4), normalbreath test (n = 17) and subjects where the diarrhea severity =constipation severity (i.e. neither predominant) (n = 15). P < 0.001between groups.

TABLE 3 Evaluation of the severity of constipation or diarrhea based onmethane production on baseline breath test. No methane Methane p-valueConstipation severity 2.3 ± 0.2 4.1 ± 0.3 <0.001 Diarrhea severity 3.0 ±0.2 1.4 ± 0.4 <0.001 C − D score* −0.7 ± 0.3  2.8 ± 0.5 <0.00001*C − D score represents the difference between severity of constipationand diarrhea. This was done to show an increased relative weight ofconstipation to diarrhea with methane excretors.

Regardless of any argument as to whether the breath test reliablydetects SIBO or not, the data in this study support a role of the LBT inIBS treatment as it is only when the subsequent LBT is normal that thegreatest symptom improvements are realized.

Although the discussion has thus far focused on the abnormal breath testrepresenting abnormal intestinal flora, another possible interpretationneed to be discussed. The abnormal breath tests seen in the study couldrepresent rapid transit. Studies have suggested that small bowel transitis accelerated in diarrhea predominant IBS (34-36). Similar studiessuggest that subjects with constipation predominant IBS have delayedtransit (34, 35). If transit is the explanation for the abnormal breathtest findings then subjects with constipation predominant IBS shouldhave delayed gas rise on breath test. On the contrary, in our study,breath tests were abnormal irrespective of subgroup of IBS suggestingthat transit alone cannot explain the findings. Furthermore, theclinical improvement in a composite score (consisting of diarrhea,constipation and abdominal pain) that depends on the normalization ofthe LBT cannot be explained on the basis of transit alone.

In summary, in this double-blind, randomized, placebo-controlled study,we found a higher prevalence of abnormal lactulose breath tests in IBSpatients than controls, indicative of SIBO. In addition, we found thatantibiotics were more effective than placebo in terms of symptomimprovement and normalization of the breath test produced an evengreater improvement of IBS symptoms, substantiating results from aprevious study (12). Furthermore, we found that methane excretion onbreath testing was highly associated with the constipation predominantsubgroup of IBS. The ability to identify subgroups of IBS based on LBTfurther supports the association between SIBO and IBS. The presence ofSIBO in IBS patients is consistent with the existence of persistentantigenic challenge in IBS.

Example 2 Administration of Methane to the Distal Gut SlowsGastrointestinal Transit

We now show that methane administered directly to the distal gutproduces a slowing of gastrointestinal transit. In dogs equipped withduodenal (10 cm from pylorus) and mid-gut (160 cm from pylorus)fistulas, intestinal transit was compared across an isolated 150 cm testsegment (between fistulas) while the proximal segment of the gut wasperfused with pH 7.0 phosphate buffer at 2 mL/min for 90 minutes. Roomair (n=three dogs) or methane (n=three dogs) was delivered into thedistal gut as a 180-ml bolus at time 0. Sixty minutes after the start ofthe perfusion, 20 μCi of ^(99 m)Tc-DTPA (diethylenetriaminepentaaceticacid) was delivered as a bolus into the proximal segment of the gut.Intestinal transit was then measured by counting the radioactivity of 1ml samples collected every 5 minutes from the diverted output of themid-gut fistula.

Intestinal transit was calculated by determining the area under thecurve (AUC) of the cumulative percent recovery of the radioactive markerin the control (air administration) and experimental (methaneadministration) dogs. The square root values of the AUC (Sqrt AUC),where 0=no recovery by 30 minutes and 47.4=theoretical, instantaneouscomplete recovery by time 0, were compared for the control andexperimental animals, using 2-way repeated measures ANOVA.

The results shown in FIG. 6, demonstrate that administration of methaneto the distal gut substantially slowed the rate of intestinal transit inthe experimental group, compared to the control.

Example 3

The following study confirmed and further investigated the relationshipbetween gastrointestinal complaints (specifically, diarrhea andconstipation) in IBS-diagnosed subjects with SIBO and gas excretion onLBT in a large prospectively collected database. The prevalence of gasexcretion patterns in IBS and the predominantly diarrheal conditions ofCrohn's disease and ulcerative colitis were also compared.

A. Materials and Methods.

Patient Population

Consecutive patients referred for a lactulose breath test (LBT) to theCedars-Sinai Medical Center, GI Motility Program from 1998-2000completed a questionnaire designed to assess bowel symptoms aspreviously described (12) after approval from the institutional reviewboard. Subjects were requested to rate the severity of nine symptoms(diarrhea, constipation, abdominal pain, bloating, sense of incompleteevacuation, straining, urgency, mucus, and gas) on a scale of 0-5, 0signifying the absence of the symptom. The questionnaire also inquiredwhether subjects had Crohn's disease (CD) or ulcerative colitis (UC). Ofsubjects reporting a history of inflammatory bowel disease (IBD), onlythose whose diagnosis had been confirmed by the Cedars-SinaiInflammatory Bowel Disease Center were included in the analysis. Thediagnosis of IBS was identified if subjects fulfilled Rome I criteria(7). Subjects found to have both IBD and IBS were assigned to the IBDsubgroup.

Subjects with conditions predisposing to rapid trait (short bowelsyndrome, gastrectomy, etc.), those taking narcotic medications, andthose without evidence of overgrowth on LBT were excluded.

Lactulose Breath Test (LBT)

After an overnight fast, subjects completed the questionnaire. Abaseline breath sample was then obtained after which subjects ingested10 g of lactulose syrup (Inalco Spa, Milano, Italy, packaged by XactdoseInc., South Beloit, Ill.). This was followed by 1 ounce of sterilewater. Breath samples were then collected every 15 minutes for 180minutes. Each sample was analyzed for hydrogen, methane, and carbondioxide gas concentration within 15 minutes of collection using a ModelSC Quintron gas chromatograph (Quintron Instrument Company, Milwaukee,Wis.). CO₂ was analyzed to correct for the quality of the alveolarsampling.

Three different abnormal gas patterns were described upon completion ofthe test:

-   1. Hydrogen positive breath test: Rise in breath hydrogen    concentration of >20 ppm within 90 minutes of lactulose ingestion    (18, 19, 37, 38).-   2. Hydrogen and methane positive breath test: Rise in both breath    hydrogen and methane concentrations of >20 ppm within 90 minutes of    lactulose ingestion.-   3. Methane positive breath test: Rise in breath methane    concentration of >20 ppm within 90 minutes of lactulose ingestion.    Data Analysis

For all subjects with SIBO, mean diarrhea and constipation severityscores among the three abnormal gas patterns were compared.

Based on symptom severity scores, the entire IBS group was furthersubdivided into diarrhea-predominant and constipation-predominantsubgroups. Constipation-predominant IBS was identified if a subject'sseverity score exceeded his or her diarrhea severity score, whereas thereverse applied for diarrhea-predominant IBS. Subjects who had aconstipation severity score equal to the diarrhea severity score(indeterminate pattern) were excluded from the IBS subgroup analysis.The percentage of IBS subjects within each abnormal gas pattern whoreported constipation-predominant or diarrhea-predominant symptoms wastabulated. The prevalence of methane production between the IBSsubgroups was also compared.

Subsequently, a mean C-D score was obtained by calculating thedifference between the constipation and diarrhea severity scores. Thiswas used to examine the relative weight of constipation to diarrhea inindividual subjects. The C-D score was compared among the three abnormalbreath gas patterns in the group as a whole and among IBS subjects.

Finally, the prevalence of each of the three abnormal gas patterns wasevaluated in subjects with CD and UC. The prevalence of methaneproduction was contrasted between subjects with IBS and IBD.

Statistical Analysis

A one-way ANOVA was conducted to compare symptom severity scores amongthe three gas patterns on LBT. Prevalance data was analyzed with achi-square test.

B. Results:

Subjects

At the time of analysis, 772 patients were referred for a LBT andentered into the database. One hundred eighty-three subjects withnegative breath tests, and 38 subjects either taking narcoticmedications or with conditions predisposing to rapid transit, wereexcluded. A total of 551 subjects remained for analysis. Of these, 78carried the diagnosis of IBD (49 with CD and 29 with UC) and 296 withoutIBD fulfilled Rome I criteria for IBS. Of the subjects with IBS, 120reported constipation-predominant symptoms, 111 had diarrhea-predominantsymptoms, and 65 had a constipation severity score equal to the diarrheaseverity score.

Bacterial Overgrowth Analysis

When the entire group of subjects with SIBO was evaluated (n=551), thediarrhea severity scores differed significantly among the three abnormalbreath test patterns (one-way ANOVA, p<0.00001) FIG. 7). Subjects whoexcreted methane reported significantly lower diarrhea severity scoresthan those who produced hydrogen only. Constipation severity alsodiffered significantly among the breath test patterns (p<0.05), withhigher severity scores reported by subjects who produced methane.

Among all IBS subjects (n=296), diarrhea severity scores also differedsimilarly (one-way ANOVA, p<0.001) with lower severity reported by thosewho produced methane than those who produced hydrogen gas alone (FIG.8).

When the C-D score was evaluated as a reflection of the degree ofconstipation with respect to diarrhea, the effect of methane was evenmore obvious (FIG. 9). In both the total group and the IBS subjects,constipation was by far the prevailing symptom in individuals, whereasdiarrhea was the prevailing symptom in subjects with only hydrogen.

When IBS subgroups were compared, constipation-predominant IBS wasreported by 91 (37%) of the hydrogen-excreting subjects, 23 (52.3%) ofthe hydrogen and methane-excreting subjects and 6 (100%) of themethane-excreting subjects. By contrast, diarrhea-predominant IBS wasobserved in 105 (42.7%) of the hydrogen excretors, 6 (13.6%) of thehydrogen and methane excretors, and none of the methane excretors FIG.10).

Inflammatory Bowel Disease and Methane

The predominant gas excreted by patients with IBD was hydrogen alone,detected in 47 of 49 subjects (95.9%) with Crohn's disease and 29 of 29(100%) subjects (100%) with ulcerative colitis. (FIG. 11).

Methane Production Between Subjects with IBS and IBD

The percentage of subjects with IBS who produced each of the three gaspatterns was tabulated. Of 296 IBS subjects, 246 (83.1%) producedhydrogen gas alone, 44 (14.9%) produced hydrogen and methane gas, and 6(2.0%) produced methane gas alone. Methane production dependedsignificantly upon whether or not subjects had IBS or IBD. IBS subjectswere more likely to produce methane gases than subjects with ulcerativecolitis or Crohn's disease (OR 7.7, CI 1.8 - 47.0, p<0.01) (Table 4).TABLE 4 Comparison of prevalence of methane to non-methane gasproduction between subjects with IBS and IBD. Disease Type CH4 Non-CH4IBS (n = 296) 50 246 UC or CD (n = 82) 2 76Chi square 9.4, OR 7.7, CI 1.8-47.0, p-value <0.01

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1. A method of manipulating the rate of gastrointestinal transit in amammalian subject, comprising: (a) increasing the rate ofgastrointestinal transit by causing the partial pressure of methane inthe subject's intestines to be decreased; and (b) decreasing the rate ofgastrointestinal transit by causing the partial pressure of methane inthe subject's intestines to be increased.
 2. The method of claim 1,wherein performing (a) is accomplished by administering to the subject'sintestinal lumen a selective inhibitor of methanogenesis.
 3. The methodof claim 1, wherein performing (a) is accomplished by administering tothe subject's intestinal lumen a methanogen-displacing probiotic agent.4. The method of claim 1, wherein performing (a) is accomplished byadministering to the subject's intestinal lumen a prebiotic agent thatinhibits the growth of methanogenic bacteria or promotes the growth ofcompeting non-methanogenic intestinal flora.
 5. The method of claim 1,wherein performing (b) is accomplished by administering methane gas tothe intestinal lumen of the subject.
 6. The method of claim 1, whereinperforming (b) is accomplished by administering a methanogenic probioticagent to the intestinal lumen of the subject.
 7. The method of claim 1,wherein performing (b) is accomplished by administering amethanogenesis-enhancing prebiotic agent to the intestinal lumen of thesubject.
 8. The method of claim 2, wherein the inhibitor is monensin. 9.Use of a selective inhibitor of methanogensis in the manufacture of amedicament for the treatment of constipation.
 10. The use of claim 9,wherein the inhibitor is monensin.
 11. Use of a methanogen-displacingprobiotic agent in the manufacture of a medicament for the treatment ofconstipation.
 12. The use of claim 11, wherein the probiotic agent isselected from the group consisting of Lactobacillus spp.,Bifodobacterium spp., and Saccharomyces species.
 13. Use of a prebioticagent that inhibits the growth of methanogenic bacteria or promotes thegrowth of competing non-methanogenic intestinal flora in the manufactureof a medicament for the treatment of constipation.
 14. Use of methane ora methane precursor in the manufacture of a medicament for the treatmentof diarrhea.
 15. Use of a methane-enhancing probiotic agent in themanufacture of a medicament for the treatment of diarrhea.
 16. Use of amethanogenesis-enhancing prebiotic agent in the manufacture of amedicament for the treatment of diarrhea.